Oral dosage composition comprising zonnula occludens toxin and a therapeutic agent for intestinal delivery

ABSTRACT

An oral dosage composition for intestinal delivery of a therapeutic agent comprising: 
     (A) a therapeutic agent selected from the group consisting of a drug compound, a biologically active peptide and a vaccine; and 
     (B) an intestinal absorption enhancing effective amount of purified Vibrio cholera zonula occludens toxin.

The development of the present invention was supported by the Universityof Maryland, Baltimore, Md.

FIELD OF THE INVENTION

The present invention relates to an oral dosage composition forintestinal delivery comprising (A) a biologically active ingredient; and(B) an intestinal absorption enhancing effective amount of zonulaoccludens toxin, as well as a method for the use of the same.

BACKGROUND OF THE INVENTION

I. Intestinal Delivery Systems

The intestinal absorption of a large number of compounds routinely usedfor the treatment of common diseases is significantly limited by theirchemical-physical characteristics. Thus, in recent years, thedevelopment of intestinal delivery systems has been one of the mostchallenging areas of research for pharmaceutical companies.

The intestinal epithelium represents the major barrier to absorption oforally administered compounds, e.g., drugs and peptides, into thesystemic circulation. This barrier is composed of a single layer ofcolumnar epithelial cells (primarily enterocytes, globet cells,endocrine cells, and paneth cells) which are joined at their apicalsurfaces by the tight junctions (hereinafter "tj" ) (Madara et al, In:Physiology of the Gastrointestinal Tract; 2nd Ed., Ed. Johnson, RavenPress, New York, pages 1251-1266 (1987)). Compounds which are presentedin the lumen can enter the blood stream through three possibleprocesses:

(i) active or facilitative transport,

(ii) passive transcellular transport, or

(iii) passive paracellular transport.

Active or facilitative transport occurs via cellular carriers, and islimited to transport of basic components of complex molecules, such asproteins and sugars, e.g., amino acids, pentoses, and exoses.

Passive transcellular transport requires partitioning of the moleculethrough both the apical and basolateral membranes. Thus, this process islimited to relatively small hydrophobic compounds (Jackson, In:Physiology of the Gastrointestinal Tract; 2nd Ed., Ed. Johnson, RavenPress, New York, pages 1597-1621 (1987)). Consequently, with theexception of those molecules which are transported by active orfacilitative mechanisms, absorption of larger and more hydrophilicmolecules is for the most part limited to the paracellular pathway.

However, studies on the use of the paracellular pathway have not beenextensively explored, mainly because of lack of information on tjstructure and function. That is, entry of molecules through theparacellular pathway is primarily restricted by the tj (Gumbiner, Am. J.Physiol., 253:C749-C758 (1987); and Madara, J. Clin. Invest.,83:1089-1094 (1989)).

In transmission electron microscopy, tj appear as an approximately 80 nmlong region at the boundary of neighboring cells in which the plasmamembranes of adjacent cells are brought into close opposition (Farquharet al, J. Cell Biol., 17:375-412 (1963)). This structure circumscribesepithelial cells immediately below the brush border (apical domain),forming a seal between epithelial cells and their neighbors. This sealrestricts diffusion of small molecules in a charge specific manner(Pappenheimer et al, J. Membrane Biol., 102:2125-2136 (1986); Madara etal, J. Cell Biol., 102:2125-2136 (1986); Claude et al, J. Cell Biol.,58:390-400 (1973); and Bakker et al, J. Membrane Biol., 11:25-35(1989)), and completely occludes molecules with molecular radii largerthen 11 Å (Madara et al, J. Cell Biol., 98:1209-1221 (1985)). Thus,considerable attention has been directed to finding ways to increaseparacellular transport by "loosening" tj.

One approach to overcoming the restriction to paracellular transport isto co-administer biologically active ingredients with absorptionenhancing agents. For the most part, the current intestinal absorptionenhancers fall within the following general classes:

(1) calcium chelators, such as citrate and EDTA; and

(2) surfactants, such as sodium dodecyl sulfate, bile salts,palmitoylcarnitine, and sodium salts of fatty acids. However, both ofthese classes of intestinal absorption enhancers have properties whichlimit their general usefulness as a means to promote absorption ofvarious molecules.

In the case of calcium chelators, Ca²⁺ depletion does not act directlyon the tj, but rather, induces global changes in the cells, includingdisruption of actin filaments, disruption of adherent junctions,diminished cell adhesion and activation of protein kinases (Citi, J.Cell Biol., 117:169-178 (1992)). Moreover, as typical formulations onlyhave access to the mucosal surface, and luminal Ca²⁺ concentration mayvary, sufficient amounts of chelators generally cannot be administeredto adequately lower Ca²⁺ levels so as to induce opening of tj in arapid, reversible and reproducible manner.

In the case of surfactants, the potential lytic nature of these agentsraises concerns regarding safety. The intestinal epithelium provides abarrier to the entry of toxins, bacteria and viruses from the ostileexterior. Hence, the possibility of exfoliation of the epithelium usingsurfactants, and potential complications arising from increasedintestinal repair, raise concerns about the use of surfactants asintestinal absorption enhancers.

Thus, there has been a desire in the art to develop intestinalabsorption enhancers which do not have the above-discussed limitations.

II. Function and Regulation of Intestinal Tight Junctions

The tj or zonula occludens (hereinafter "ZO") are one of the hallmarksof absorptive and secretory epithelia (Madara, J. Clin. Invest.,83:1089-1094 (1989); and Madara, Textbook of Secretory Diarrhea Eds.Lebenthal et al, Chapter 11, pages 125-138 (1990). As a barrier betweenapical and basolateral compartments, they selectively regulate thepassive diffusion of ions and water-soluble solutes through theparacellular pathway (Gumbiner, Am. J. Physiol., 253 (Cell Physiol.22):C749-C758 (1987)). This barrier maintains any gradient generated bythe activity of pathways associated with the transcellular route(Diamond, Physiologist, 20:10-18 (1977)).

Variations in transepithelial conductance can usually be attributed tochanges in the permeability of the paracellular pathway, since theresistances of enterocyte plasma membranes are relatively high (Madara,supra). The ZO represents the major barrier in this paracellularpathway, and the electrical resistance of epithelial tissues seems todepend on the number of transmembrane protein strands, and theircomplexity in the ZO, as observed by freeze-fracture electron microscopy(Madara et al, J. Cell Biol., 101:2124-2133 (1985)).

There is abundant evidence that ZO, once regarded as static structures,are in fact dynamic and readily adapt to a variety of developmental(Magnuson et al, Dev. Biol., 67:214-224 (1978); Revel et al, Cold SpringHarbor Symp. Quant. Biol., 40:443-455 (1976); and Schneeberger et al, J.Cell Sci., 32:307-324 (1978)), physiological (Gilula et al, Dev. Biol.,50:142-168 (1976); Madara et al, J. Membr. Biol., 100:149-164 (1987);Mazariegos et al, J. Cell Biol., 98:1865-1877 (1984); and Sardet et al,J. Cell Biol., 80:96-117 (1979)), and pathological (Milks et al, J. CellBiol., 103:2729-2738 (1986); Nash et al, Lab. Invest., 59:531-537(1988); and Shasby et al, Am. J. Physiol., 255(Cell Physiol.,24):C781-C788 (1988)) circumstances. The regulatory mechanisms thatunderlie this adaptation are still not completely understood. However,it is clear that, in the presence of Ca²⁺, assembly of the ZO is theresult of cellular interactions that trigger a complex cascade ofbiochemical events that ultimately lead to the formation and modulationof an organized network of ZO elements, the composition of which hasbeen only partially characterized (Diamond, Physiologist, 20:10-18(1977)). A candidate for the transmembrane protein strands, occludin,has recently been identified (Furuse et al, J. Membr. Biol., 87:141-150(1985)).

Six proteins have been identified in a cytoplasmic submembranous plaqueunderlying membrane contacts, but their function remains to beestablished (Diamond, supra). ZO-1 and ZO-2 exist as a heterodimer(Gumbiner et al, Proc. Natl. Acad. Sci., USA, 88:3460-3464 (1991)) in adetergent-stable complex with an uncharacterized 130 kD protein (ZO-3).Most immunoelectron microscopic studies have localized ZO-1 to preciselybeneath membrane contacts (Stevenson et al, Molec. Cell Biochem.,83:129-145 (1988)). Two other proteins, cingulin (Citi et al, Nature(London), 333:272-275 (1988)) and the 7H6 antigen (Zhong et al, J. CellBiol., 120:477-483 (1993)) are localized further from the membrane andhave not yet been cloned. Rab 13, a small GTP binding protein has alsorecently been localized to the junction region (Zahraoui et al, J. CellBiol., 124:101-115 (1994)). Other small GTP-binding proteins are knownto regulate the cortical cytoskeleton, i.e., rho regulatesactin-membrane attachment in focal contacts (Ridley et al, Cell,70:389-399 (1992)), and rac regulates growth factor-induced membraneruffling (Ridley et al, Cell, 70:401-410 (1992)). Based on the analogywith the known functions of plaque proteins in the better characterizedcell junctions, focal contacts (Guan et al, Nature, 358:690-692 (1992)),and adherens junctions (Tsukita et al, J. Cell Biol., 123:1049-1053(1993)), it has been hypothesize that tj-associated plaque proteins areinvolved in transducing signals in both directions across the cellmembrane, and in regulating links to the cortical actin cytoskeleton.

To meet the many diverse physiological and pathological challenges towhich epithelia are subjected, the ZO must be capable of rapid andcoordinated responses that require the presence of a complex regulatorysystem. The precise characterization of the mechanisms involved in theassembly and regulation of the ZO is an area of current activeinvestigation.

There is now a body of evidence that tj structural and functionallinkages exist between the actin cytoskeleton and the tj complex ofabsorptive cells (Gumbiner et al, supra; Madara et al, supra; andDrenchahn et al, J. Cell Biol., 107:1037-1048 (1988)). The actincytoskeleton is composed of a complicated meshwork of microfilamentswhose precise geometry is regulated by a large cadre of actin-bindingproteins. An example of how the state of phosphorylation of anactin-binding protein might regulate cytoskeletal linking to the cellplasma membrane is the myristoylated alanine-rich C kinase substrate(hereinafter "MARCKS"). MARCKS is a specific protein kinase C(hereinafter "PKC") substrate that is associated with the cytoplasmicface of the plasma membrane (Aderem, Elsevier Sci. Pub. (UK), pages438-443 (1992)). In its non-phosphorylated form, MARCKS crosslinks tothe membrane actin. Thus, it is likely that the actin meshworkassociated with the membrane via MARCKS is relatively rigid (Hartwig etal, Nature, 356:618-622 (1992)). Activated PKC phosphorylates MARCKS,which is released from the membrane (Rosen et al, J. Exp. Med.,172:1211-1215 (1990); and Thelen et al, Nature, 351:320-322 (1991)). Theactin linked to MARCKS is likely to be spatially separated from themembrane and be more plastic. When MARCKS is dephosphorylated, itreturns to the membrane where it once again crosslinks actin (Hartwig etal, supra; and Thelen et al, supra). These data suggest that the F-actinnetwork may be rearranged by a PKC-dependent phosphorylation processthat involves actin-binding proteins (MARCKS being one of them).

A variety of intracellular mediators have been shown to alter tjfunction and/or structure. Tight junctions of amphibian gallbladder(Duffey et al, Nature, 204:451-452 (1981)), and both goldfish (Bakker etal, Am. J. Physiol., 246:G213-G217 (1984)) and flounder (Krasney et al,Fed. Proc., 42:1100 (1983)) intestine, display enhanced resistance topassive ion flow as intracellular cAMP is elevated. Also, exposure ofamphibian gallbladder to Ca²⁺ ionophore appears to enhance tjresistance, and induce alterations in tj structure (Palant et al, Am. J.Physiol., 245:C203-C212 (1983)). Further, activation of PKC by phorbolesters increases paracellular permeability both in kidney (Ellis et al,C. Am. J. Physiol., 263 (Renal Fluid Electrolyte Physiol. 32):F293-F300(1992)), and intestinal (Stenson et al, C. Am. J. Physiol.,265(Gastrointest. Liver Physiol., 28):G955-G962 (1993)) epithelial celllines.

III. Zonula Occludens Toxin

Most Vibrio cholerae vaccine candidates constructed by deleting the ctxAgene encoding cholera toxin (CT) are able to elicit high antibodyresponses, but more than one-half of the vaccines still develop milddiarrhea (Levine et al, Infect. Immun., 56(1):161-167 (1988)). Given themagnitude of the diarrhea induced in the absence of CT, it washypothesized that V. cholerae produce other enterotoxigenic factors,which are still present in strains deleted of the ctxA sequence (Levineet al, supra). As a result, a second toxin, zonula occludens toxin(hereinafter "ZOT") elaborated by V. cholerae and which contribute tothe residual diarrhea, was discovered (Fasano et al, Proc. Nat. Acad.Sci., USA, 8:5242-5246 (1991)). The zot gene is located immediatelyadjacent to the ctx genes. The high percent concurrence of the zot genewith the ctx genes among V. cholerae strains (Johnson et al, J. Clin.Microb., 31/3:732-733 (1993); and Karasawa et al, FEBS MicrobiologyLetters, 106:143-146 (1993)) suggests a possible synergistic role of ZOTin the causation of acute dehydrating diarrhea typical of cholera.Recently, the zot gene has also been identified in other entericpathogens ( Tschape, 2nd Asian-Pacific Symposium on Typhoid fever andother Salomellosis, 47(Abstr.) (1994)).

It has been previously found that, when tested on rabbit ileal mucosa,ZOT increases the intestinal permeability by modulating the structure ofintercellular tj (Fasano et al, supra). It has been found that as aconsequence of modification of the paracellular pathway, the intestinalmucosa becomes more permeable. It also was found that ZOT does notaffect Na⁺ -glucose coupled active transport, is not cytotoxic, andfails to completely abolish transetransepithelial resistance (Fasano etal, supra).

In the present invention, it has been demonstrated, for the first time,that ZOT induces a reversible increase in tissue permeability ofmolecules of different size and structure, and that therefore ZOT, whenco-administered with a biologically active ingredient, is able toenhance intestinal absorption of the biologically active ingredients.

SUMMARY OF THE INVENTION

An object of the present invention is to provide intestinal absorptionenhancers which rapidly open tj in a reversible and reproducible manner.

Another object of the present invention is to provide intestinalabsorption enhancers which can be used safely without damaging theintestinal epithelium.

Still another object of the present invention is to provide an oraldosage composition which allows for the systemic delivery ofbiologically active ingredients.

Yet another object of the present invention is to provide a method fororal delivery of biologically active ingredients such that they areabsorbed by the intestine.

These and other objects of the present invention, which will be apparentfrom the detailed description of the invention provided hereinafter,have been met in one embodiment by an oral dosage composition forintestinal delivery comprising:

(A) a biologically active ingredient; and

(B) an intestinal absorption enhancing effective amount of zonulaoccludens toxin.

In another embodiment, the present invention the above-described objectsof the present invention have been met by method for intestinal deliveryof a biologically active ingredient comprising orally administering anoral dosage composition for intestinal delivery comprising:

(A) a biologically active ingredient; and

(B) an intestinal absorption enhancing effective amount of zonulaoccludens toxin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of the PKC inhibitor CGP41251, and its inactiveanalogue CGP42700, on changes in tissue resistance (Rt) induced by pZ14supernatant in rabbit ileum.

FIGS. 2A and 2B show the effect of ZOT on PKC activity over time incytosolic (FIG. 2A) and membranous (FIG. 2B) subfractions.

FIG. 3 shows a dose-response curve for purified ZOT on Rt in rabbitileum.

FIG. 4 shows the effect of purified MBP-ZOT (Δ) as compared to purifiedZOT (□), and MBP control (▪) on Rt in rabbit ileum.

FIGS. 5A and 5B show the reversible effect of purified MBP-ZOT ontransepithelial transport of insulin (FIG. 5A) and immunoglobulin IgG(FIG. 5B) in rabbit ileum.

FIGS. 6A and 6B show the reversible effect of purified ZOT ontransepithelial transport of insulin (FIG. 6A) and immunoglobulin IgG(FIG. 6B) in rabbit ileum.

FIGS. 7A-7C show the effect of purified ZOT on water (□) and insulintransport (▴), as determined by an in vivo perfusion assay, in rabbitjejunum (FIG. 7A), ileum (FIG. 7B), and colon (FIG. 7C).

FIG. 8 shows the serum concentration of insulin (open bar) and ¹⁴C-PEG-4000 (shaded bar) in the absence or presence of ZOT in themesenteric vein draining a perfused segment of rabbit jejunum, ileum andcolon.

FIGS. 9A-9C show the effect of purified ZOT on water (□) andimmunoglobulin IgG transport (∘), as determined by an in vivo perfusionassay, in rabbit jejunum (FIG. 9A), ileum (FIG. 9B), and colon (FIG.9C).

FIG. 10 shows the serum concentration of immunoglobulin (open bar) and¹⁴ C-PEG4000 (shaded bar) in the absence or presence of ZOT in themesenteric vein draining a perfused segment of rabbit jejunum, ileum andcolon.

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, in one embodiment, the present invention relates toan oral dosage composition for intestinal delivery comprising:

(A) a biologically active ingredient; and

(B) an intestinal absorption enhancing effective amount of zonulaoccludens toxin.

Oral dosage compositions for small intestinal delivery, e.g., smallintestinal delivery, are well-known in the art. Such oral dosagescompositions generally comprise gastroresistent tablets or capsules(Remington's Pharmaceutical Sciences, 16th Ed., Eds. Osol, MackPublishing Co., Chapter 89 (1980); Digenis et al, J. Pharm. Sci.,83:915-921 (1994); Vantini et al, Clinica Terapeutica, 145:445-451(1993); Yoshitomi et al, Chem. Pharm. Bull., 40:1902-1905 (1992); Thomaet al, Pharmazie, 46:331-336 (1991); Morishita et al, Drug Design andDelivery, 7:309-319 (1991); and Lin et al, Pharmaceutical Res.,8:919-924 (1991)); each of which is incorporated by reference herein inits entirety).

Tablets are made gastroresistent by the addition of, e.g., eithercellulose acetate phthalate or cellulose acetate terephthalate.

Capsules are solid dosage forms in which the biologically activeingredient(s) is enclosed in either a hard or soft, soluble container orshell of gelatin. The gelatin used in the manufacture of capsules isobtained from collagenous material by hydrolysis. There are two types ofgelatin. Type A, derived from pork skins by acid processing, and Type B,obtained from bones and animal skins by alkaline processing. The use ofhard gelatin capsules permit a choice in prescribing a singlebiologically active ingredient or a combination thereof at the exactdosage level considered best for the individual subject. The hardgelatin capsule consists of two sections, one slipping over the other,thus completely surrounding the biologically active ingredient. Thesecapsules are filled by introducing the biologically active ingredient,or gastroresistent beads containing the biologically active ingredient,into the longer end of the capsule, and then slipping on the cap. Hardgelatin capsules are made largely from gelatin, FD&C colorants, andsometimes an opacifying agent, such as titanium dioxide. The USP permitsthe gelatin for this purpose to contain 0.15% (w/v) sulfur dioxide toprevent decomposition during manufacture.

The particular biologically active ingredient employed is not criticalto the present invention, and can be, e.g., any drug compound,biologically active peptide, vaccine, or any other moiety otherwise notabsorbed through the transcellular pathway, regardless of size orcharge.

Examples of drug compounds which can be employed in the presentinvention include drugs which act on the cardiovascular system, drugswhich act on the central nervous system, antineoplastic drugs andantibiotics.

Examples of drugs which act on the cardiovascular system which can beemployed in the present invention include lidocaine, adenosine,dobutamine, dopamine, epinephrine, norepinephrine and phentolamine.

Examples of drugs which act on the central nervous system which can beemployed in the present invention include doxapram, alfentanil, dezocin,nalbuphine, buprenorphine, naloxone, ketorolac, midazolam, propofol,metacurine, mivacurium and succinylcholine.

Examples of antineoplastic drugs which can be employed in the presentinclude cytarabine, mitomycin, doxorubicin, vincristine and vinblastine.

Examples of antibiotics which can be employed in the present includemethicillin, mezlocillin, piperacillin, cetoxitin, cefonicid,cefmetazole and aztreonam.

Examples of biologically active peptides which can be employed in thepresent invention include hormones, lymphokines, globulins, andalbumins.

Examples of hormones which can be employed in the present inventioninclude testosterone, nandrolene, menotropins, insulin andurofolltropin.

Examples of lymphokines which can be employed in the present inventioninclude interferon-α, interferon-β, interferon-γ, interleukin-1,interleukin-2, interleukin-4 and interleukin-8.

Examples of globulins which can be employed in the present inventioninclude α-globulins, β-globulins and γ-globulins (immunoglobulin).

Examples of immunoglobulins which can be employed in the presentinvention include polyvalent IgG or specific IgG, IgA and IgM, e.g.,anti-tetanus antibodies.

An example of albumin which can be employed in the present invention ishuman serum albumin.

Examples of vaccines which can be employed in the present inventioninclude peptide antigens and attenuated microorganisms and viruses.

Examples of peptide antigens which can be employed in the presentinvention include the B subunit of the heat-labile enterotoxin ofenterotoxigenic E. coli, the B subunit of cholera toxin, capsularantigens of enteric pathogens, fimbriae or pili of enteric pathogens,HIV surface antigens, dust allergens and acari allergens.

Examples of attenuated microorganisms and viruses which can be employedin the present invention include those of enterotoxigenic Escherichiacoli, enteropathogenic Escherichia coli, Vibrio cholerae, Shigellaflexneri, Salmonella typhi and rotavirus (Fasano et al, In: LeVaccinazioni in Pediatria, Eds. Vierucci et al, CSH, Milan, pages109-121 (1991); Guandalini et al, In: Management of Digestive and LiverDisorders in Infants and Children, Elsevior, Eds. Butz et al, Amsterdam,Chapter 25 (1993); Levine et al, Sem. Ped. Infect. Dis., 5:243-250(1994); and Kaper et al, Clin. Micrbiol. Rev., 8:48-86 (1995), each ofwhich is incorporated by reference herein in its entirety).

The amount of biologically active ingredient employed is not critical tothe present invention and will vary depending upon the particularingredient selected, the disease or condition being treated, as well asthe age, weight and sex of the subject being treated.

The amount of zonula occludens toxin (hereinafter "ZOT") employed isalso not critical to the present invention and will vary depending uponthe age, weight and sex of the subject being treated. Generally, thefinal concentration of ZOT employed in the present invention to enhanceabsorption of the biologically active ingredient by the intestine is inthe range of about 10⁻⁵ M to 10⁻¹⁰ M, preferably about 10⁻⁶ M to5.0×10⁻⁵ M. To achieve such a final concentration in the intestine, theamount of ZOT in a single oral dosage composition of the presentinvention will generally be about 40 ng to 1000 ng, preferably about 400ng to 800 ng.

The ratio of biologically active ingredient to ZOT employed is notcritical to the present invention and will vary depending upon theamount of biologically active ingredient to be delivered within theselected period of time. Generally, the weight ratio of biologicallyactive ingredient to ZOT employed in the present invention is in therange of about 1:10 to 3:1, preferably about 1:5 to 2:1.

ZOT is produced by V. cholerae. The particular strain of V. cholera fromwhich ZOT is derived is not critical to the present invention. Examplesof such V. cholerae strains include strain 569B, 395 and E7946 (Levineet al, supra; Johnson et al, supra; and Karasawa et al, supra).

As used herein, "ZOT" refers to the mature protein of 399 amino acids,as well as mutants thereof which retain the ability to regulate tj. Forexample, an N-terminal deletion of amino acids 1-8 can be made withouteffecting ZOT activity, and N-terminal fusion proteins of ZOT can bemade without effecting ZOT activity. Such mutants can be readilyprepared by site-directed mutagenesis, and screened for ZOT activity asdescribed herein.

ZOT can be obtained and purified, e.g., by genetically-engineered E.coli strains overexpressing the zot gene (Baudry et al, Infect. Immun.,60:428-434 (1992)), alone or fused to other genes, such as maltosebinding protein (see Example 5 below) or glutathione-S-transferase (seeExample 6 below).

The following examples are provided for illustrative purposes only, andare in no way intended to limit the scope of the present invention.

EXAMPLE 1 Effect of ZOT on Enterocyte Cytoskeleton

In the small intestine (Madara et al, J. Cell Biol., 97:125-136 (1983))and in renal cells (Meza et al, J. Cell Biol., 87:746-754 (1980)),pathophysiological alterations in cytoskeletal arrangement may producemajor alterations in occluding tj resistance, charge selectivity, andstructure. The response elicited by these specific perturbationsconsists of expansion of occluding tj structure, and changes inparacellular cation selectivity. These data suggest that theparacellular pathway may be regulated by intracellular events whichproduce phenotypic alterations in the cell surface structure thatregulates ZO. However, the cascade of intracellular events leading tosuch tj modifications is still poorly defined.

A. The Effect of ZOT on Cytoskeletal Arrangement

To evaluate whether ZOT has an effect on cytoskeletal rearrangement, theM_(r) >10,000 supernatant fraction obtained after culturing V. choleraestrain CVD110 transformed with plasmid pZ14 (hereinafter "pZ14supernatant"), was tested on tissue cultures of IEC6 cells.

CVD110is a V. cholerae (El Tor biotype) strain in which all known toxingenes (ctxA, zot and ace genes) have been deleted (Michalski et al,Infect. Immun., G1:4462-4468 (1993)).

Plasmid pZ14 contains the zot gene transcribed by the inducible tacpromoter. Plasmid pZ14 was constructed by digesting pBB241 with HindIII.pBB241 was obtained by cloning a ClaI-XbaI fragment containing theentire zot sequence into plasmid pUC19 (Baudry et al, supra). The 5'overhang was filled in with Klenow fragment, and the linearized plasmidwas digested with XbaI, yielding a zot fragment of 1.5 kb. This fragmentwas cloned into vector pTTQ181 (Amersham, Arlington Heights, Ill. )which was modified by interruption of the Amp^(R) gene by the Kan^(R)cassette found in pHSG274 described in Maniatis et al, MolecularCloning, A Laboratory Manual, Cold Spring Harbor (1989). That is,pTTQ181 was digested with EcoRI, filled in, and digested with XbaI. The1.5 kb XbaI zot fragment was ligated into the resulting vector in thecorrect orientation, and was designated "pZ14".

The IEC6 cells were derived from crypt cells of germ-free rat smallintestine (Quaroni et al, In: Methods in Cell Biology, Chapter 20,21B:403-426 (1980)), and were grown in complete medium in cell-cultureflasks (Falcon) at 37° C. in an atmosphere of 95% O₂ /5% CO₂. Thecomplete medium comprised Dulbecco's modified Eagle medium supplementedwith 4.5 g/l glucose, and containing 5.0% (v/v) fetal bovine serum(irradiated), 10 μg/ml insulin, 4.0 mM L-glutamine, 50 U/ml penicillin,and 50 μg/ml streptomycin. The passage number used varied from between15 and 20.

The M_(r) >10,000 supernatant fraction was prepared as follows. CVD110transformed with pZ14 was cultured overnight at 37° C., in Luria Bertani(hereinafter "LB") broth containing 50 μg/ml kanamycin so as to selectkanamycin-resistant strains harboring pZ14 plasmid. The cultures werethen diluted to obtain an initial OD 600 nm of 0.4-0.5. Next, to induceexpression of ZOT from the tac promoter, 2.0 mM ofIsopropyl-Thio-β-D-Galactopyranoside (IPTG) (5'-3' Incorporation,Boulder, Colo.), was added to the cultures, which were incubated at 37°C. for another 2 hr. Next, the culture medium was collected, cooled andcentrifuged at 5,000×g. for 10 min at 40° C. The resulting liquid wascollected and passed through a 0.45 μm filter (Millipore). The resultingculture supernatant was then subjected to ultrafiltration throughCentricon filters (Vangard International Corp., N.J.) with a 10 kDa Mcut-off size. The M_(r) >10 kDa fraction was washed twice with phosphatebuffered saline (pH 7.4) (hereinafter "PBS"), reconstituted to theoriginal volume in PBS, and tested for activity on cytoskeletalrearrangement as discussed below.

1. Fluorescent Microscopy

2.0×10⁴ IEC6 cells/ml were seeded onto 13 mm diameter glass coverslips,and exposed to 30 μl of the pZ14 supernatant (1:33 dilution) or to 30 μlof PBS as a negative control, for 24 hr at 37° C. The cells were thenfixed in 3.7% (v/v) formaldehyde in PBS (pH 7.4) for 10 min at roomtemperature. After washing in the same buffer, the cells werepermeabilized with 0.5% (v/v) Triton X-100 (Sigma) in PBS (pH 7.4) for10 min at room temperature. The resulting fixed cells were thenincubated with 5.0 μg/ml fluorescein-phalloidin (Sigma) at 37° C. for 30min. Finally, the cells were washed with PBS, the coverslips weremounted with 1:1 glycerol-PBS (pH 8.0), and 200 cells for each conditionwere analyzed in triplicate in blind fashion with a Nikon Optiphotfluorescence microscope so to visualize the F-actin cytoskeletal network(defined as stress fibers) of the cells.

The normal distribution of stress fibers was seen within the cellsincubated with the PBS negative control. However, when the IEC6 cellswere incubated with pZ14 supernatant, a complete rearrangement of thecytoskeleton was observed (peaking at 24 hr incubation), with the stressfibers redistributed to the cell periphery.

After 48 hr exposure to pZ14 culture supernatant, F-actin organizationremained unchanged from the cells exposed for 24 hr. F-actinorganization in the negative controls remained unchanged over the 48 hrstudy period.

2. Scanning Electron Microscopy

In parallel studies, the IEC6 cells incubated with pZ14 supernatant orPBS negative control were analyzed by scanning electron microscopy.

More specifically, IEC6 cells were seeded at 5.0×10⁹ cells/well onto 13mm diameter glass coverslips inserted in the wells of 24-well plates.After 24 hr exposure to either 30 μl of pZ14 supernatant or 30 μl of PBSnegative control at 37° C., the cells were fixed with 2.5% (v/v)glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 20 min at roomtemperature. Following post-fixation in 1.0% OsO₄ (w/v) for 30 min, thecells were dehydrated through graded ethanols, critical point driedunder CO₂, and gold coated by sputtering. The samples were examined witha Cambridge scanning electron microscope.

Modifications of the surface of ZOT-exposed cells were seen whencompared to the PBS negative control. That is, ZOT mainly induced a lossof microvilli around the cell periphery with central sparing. Thesesurface changes are consistent with the F-actin redistribution inducedby ZOT seen in the fluorescent microscopy above.

3. Rabbit Ileum Studies

Experiments on cytoskeletal reorganization were then performed usingrabbit ileum mounted in Using chambers as described by Fasano et al,Proc. Nat. Acad. Sci., USA, 8:5242-5246 (1991).

More specifically, 2-3 kg adult male New Zealand white rabbits weresacrificed by cervical dislocation. A 20 cm segment of ileum wasremoved, rinsed free of the intestinal content, opened along themesenteric border, and stripped of muscular and serosal layers. Eightsheets of mucosa so prepared were then mounted in lucite Ussing chambers(1.12 cm² opening), connected to a voltage clamp apparatus (EVC 4000WPI, Saratosa, Fla.), and bathed with freshly prepared Ringer's solutioncomprising 53 mM NaCl, 5.0 mM KCl, 30.5 mM mannitol, 1.69 mM Na₂ HPO₄,0.3 mM NaH₂ PO₄, 1.25 mM CaCl₂, 1.1 mM MgCl₂, and 25 mM NaHCO₃. Thebathing solution was maintained at 37° C. with water-jacketed reservoirsconnected to a constant-temperature circulating pump and gassed with 95%O₂ /5% CO₂.

300 μl of pZ14 supernatant was added to the mucosal side. 300 μl of pZ14supernatant was also added to the serosal side to preserve the osmoticbalance. The potential difference (PD) was measured every 10 min, andthe short-circuit current (Isc) and tissue resistance (Rt) werecalculated as described by Fasano et al, supra. Because of tissuevariability, data were calculated as ΔRt (Rt at time x)-(Rt at time 0).At the end of every experiment, 0.5 mM glucose was added to the mucosalside of each chamber. Only those tissues which showed an increase in Iscin response to glucose (indicating tissue viability) were included inthe analysis.

Once the maximum effect of ZOT on Rt was obtained after 60 min ofincubation, the tissues exposed to either the pZ14 supernatant or thePBS negative control were fixed, and stained for F-actin withfluorescein-phalloidin, as described above.

Control rabbit ileum was characterized by homogeneous fluorescentstaining of the brush border, and a columnar distribution of actinmicrofilaments between adjacent enterocytes. Rabbit ileum exposed topZ14 supernatant showed an irregular staining of the brush border,associated with a partial redistribution of F-actin within theunderlying cytoplasm. These changes were more evident in the maturecells of the tip of the villi as compared to the less differentiatedcrypt cells.

4. Evaluation of Actin Pools

Actin is one of the principal constituents of the cellular cytoskeletalnetwork, and shifts between the soluble monomeric G-actin pool andfilamentous F-actin pool reflects changes in actin organization(Drenchahn et al, J. Cell Biol., 107:1037-1048). Thus, the effect of ZOTon G- and F-actin pools was evaluated.

More specifically, bovine pulmonary artery endothelial cells (Goldblumet al, J. Cell Physiol., 157:197-205 (1993)), obtained from the AmericanType Culture Collection (Rockville, Md.) ATCC No. 209CCL, were grown at37° C. under 95% O₂ /5% CO₂ in Dulbecco's modified Eagle's mediumenriched with 20% (v/v) heat-inactivated (560° C., for 30 min) fetalcalf serum (HyClone Laboratories, Logan UT), 4.0 mM L-glutamine,non-essential amino acids, and vitamins in the presence of 50 U/mlpenicillin and 50 μg/ml streptomycin. The cultures were determined to beendothelial by uniform morphology and by quantitative determination ofangiotensin-converting enzyme activity with commercially available ³H-benzyl-Phe-Ala-Pro substrate (Ventrex Laboratories, Inc., Portland,Me.). Only cell passages 3-7 were used.

Endothelial F-actin was fluorometrically measured as described byGoldblum et al, supra. More specifically, 5.8×10⁵ endothelial cells in2.0 ml of Dulbecco's modified Eagle's medium enriched with 20% (v/v)heat-inactivated (56° C., 30 min) fetal calf serum (HyCloneLaboratories, Logan, Utah), 4.0 mM L-glutamine, nonessential aminoacids, and vitamins in the presence of 50 U/ml penicillin and 50 μg/mlof streptomycin, were seeded into the wells of 6-well plates andcultured for 72 hr at 37° C. in 95% O₂ /5% CO₂. The monolayers wereexposed to 30 μl of pZ14 supernatant (1:33 dilution) for 24 hr, afterwhich they were washed twice in a buffer comprising 75 mM KCl, 3.0 mMMgSO₄, 1.0 mM ethylene glycol tetraacetic acid (EGTA), 10 mM imidazole,0.2 mM dithiothreitol (DTT), 10 μg/ml aprotinin, and 0.1 mMphenylmethylsulfonyl fluoride (PMSF), and fixed with 3.7% (v/v)formaldehyde for 15 min. Next, the monolayers were permeabilized with0.2% (v/v) Triton X-100 in the above buffer for 5 min at roomtemperature, stained with 1.65×10⁻⁷ M NBD-phallicidin (Sigma) for 20min, and extracted with ice cold methanol overnight at -20° C. Stainingand extractions were performed in the dark. Extracts were harvested intocurvettes and intraendothelial fluorescence was measured in aPerkin-Elmer LS30 luminescence spectrometer at room temperature at 465nm excitation (10 nm slit) and 535 nm emission (10 nm slit), andexpressed in arbitrary fluorescent units per mg total endothelial cellprotein.

Application of NBD-phallicidin to fixed permeabilized endothelialmonolayers resulted in 78.8% penetration of the probe into the cell, and99.98% of intracellular probe was extracted with a single methanoltreatment, as measured by fluorometry.

Actin reorganization can involve reciprocal changes in the solublemonomeric G- and filamentous F-actin pool, while the total actin poolremains unchanged. The above-described methodologies for determining F-and G-actin pools involve fixation, permeabilization, and extractionprocedures which preclude protein determinations on the same bovinepulmonary artery endothelial monolayers. Therefore, for standardizationof F- and G-actin measurements, additional simultaneously plated cellsfor total protein determination were cultured under identical conditionsas the monolayers assayed for the F- and G-actin pools.

More specifically, post-confluent bovine pulmonary artery endothelialmonolayers in the wells of 6-well plates were washed twice with PBS (pH7.4) and gently detached by exposure to 0.5 mg/ml trypsin for 1-2 minwith gentle agitation, followed immediately by neutralization with theDulbecco's medium described above containing PBS. The cells werecentrifuged at 600 ×g. for 10 min at 4° C., again washed twice with PBS(pH 7.4), and lysed in lysing buffer comprising 3.0% (w/v) sodiumdodecyl sulfate (SDS), 1.0 mM DTT, 10 mM PMSF, 1.0 mM ethylenediaminetetraacetic acid (EDTA), and 50 mM Tris-HCl (pH 8.0). The lysates werethen assayed for protein concentration using the standard Bio-Rad DCProtein Assay (Bio-Rad Chemical Division, Richmond, Calif.)

Endothelial G-actin was measured using the DNAseI inhibition assay asdescribed by Goldblum et al, supra. More specifically, bovine pancreasDNAseI (Sigma) was dissolved in 0.125 M Tris-HCl (pH 7.5) 5.0 mM MgCl₂,2.0 mM CaCl₂, 1.0 mM NaN₃, and 0.1 mM PMSF at a concentration of 10mg/ml so as to increase its stability. The stock solution was thendiluted 100x with 20 mM imidazole (pH 7.5), 30 mM NaCl₂, and 15% (v/v)glycerol. The enzyme was freshly made every 2 hr and kept on ice. Calfthymus DNA (type 1, Sigma) was used as substrate for the DNAseI. Thefibrous DNA preparation was cut into fine pieces and suspended in 0.1 MTris-HCl (pH 7.5), 4.0 mM MgSO₄, 1.8 mM CaCl₂ at a concentration of 80mg DNA/ml. The DNA was brought into solution by slow stirring at 4° C.for 48 hr, after which the solution was sequentially passed through 0.45μm and 0.22 μm pore size filters, and stored at 4° C. The absorbance ofthe final substrate at 260 nm varied between 1.05 and 1.15. The DNAseIwas then mixed with DNA substrate in the cuvette of a Gilford responsespectrophotometer (Ciba Corning Diagnostics), and the slope of thelinear portion of the OD at 260 nm recorded. Purified bovine skeletalmuscle actin (Sigma) dissolved in 20 mM Tris-HCl (pH 7.5), 1.0 M Naacetate, 1.0 mM CaCl₃, 1.0 mM adenosine triphosphate (ATP), 0.75 Mguanidine-HCl was used as the G-actin standard to calibrate the assay.

Bovine endothelial monolayers grown in 6-well plates were exposed to 30μl of pZ14 supernatant. The monolayers were washed with Dulbecco's PBSwithout Ca²⁺ and Mg²⁺, and permeabilized with 0.5 ml/well of lysingbuffer comprising Hanks Balanced Salt Solution containing 1.0% (v/v)Triton X-100, 2.0 mM MgCl₃, 2.0 mM EGTA, 0.2 mM ATP, 0.5 mM DTT, 0.1 mMPMSF for 5 min. The G-actin-containing supernatants then were tested inthe DNAseI inhibition assay to generate inhibitory activities that fellon the linear portion of the standard curve, i.e., 30-70% inhibition.The inhibitory activities were interpolated to G-actin concentrations,which were used to calculate G-actin expressed in μg/mg totalendothelial cell protein.

Monolayers exposed to 30 μl of pZ14 supernatant boiled for 15 min, so asabolish the effect of ZOT on Rt, and 30 μl of supernatant from V.cholera strain CVD110 (Michalski et al, supra) transformed with pTTQ181(Amersham, Arlington Heights, Ill.) (hereinafter "pTTQ181 supernatant")were used as negative controls.

pZ14 supernatant induced a significant decrease in the G-actin pool(-27%), and a reciprocal increase in the F-actin pool (+22%) as comparedto either pTTQ181 supernatant or pZ14-boiled supernatants (see Table Ibelow). This G- to F-actin shift is compatible with actinpolymerization. This effect was completely abolished by boiling the pZ14supernatant for 15 min.

                                      TABLE I                                     __________________________________________________________________________    G- and F-Actin Assays and Effect of PKC                                       Inhibitor CGP41251 on Actin Polymerization                                               G-Actin       F-Actin                                              Strain (N)*                                                                              (μg/mg protein)                                                                     p value**                                                                          (U/mg protein)                                                                         p value**                                   __________________________________________________________________________    pTTQ181 (20)                                                                             106.1                                                                             (3.92)***                                                                          --   341.34                                                                            (5.28)***                                                                          --                                          pZ14 (38)  76.0                                                                              (4.16)                                                                             0.0001                                                                             415.73                                                                            (22.03)                                                                            0.0036                                      CGP41251 + pZ14 (30)                                                                     108.6                                                                             (7.2)                                                                              0.7803                                                                             335.67                                                                            (12.1)                                                                             0.673                                       CGP42700 + pZ14 (30)                                                                     84.8                                                                              (4.0)                                                                              0.0011                                                                             446.17                                                                            (18.5)                                                                             0.0001                                      __________________________________________________________________________     *Number of experiments                                                        **compared to pTTQ181 negative control                                        ***Standard Error (S.E.)                                                 

5. Effect of ZOT on F-actin and ZO-1 Protein Distribution

Parallel studies were conducted on the effect of ZOT on F-actin and ZO-1protein distribution in IEC6 cells apically sectioned to include the tjcomplex.

More specifically, 2.0×10⁴ IEC6 cells/ml cultured on coverslips wereincubated at 37° C. for 24 hr with 30 μl of pZ14 supernatant. After theincubation, the cells were fixed in 3.7% (v/v) formaldehyde in PBS (pH7.4) for 10 min at room temperature. After washing in the same buffer,the cells were permeabilized with 0.5% (v/v) Triton X-100 in PBS (pH7.4) for 10 min at room temperature. The cells were then incubated withboth 5.0 μg/ml fluorescein-phalloidin and 2.0 μg/ml anti-ZO-1 antibody7445 (Zymed laboratory Inc, South San Francisco, Calif.) at 37° C. for30 min. Finally, the cells were washed with PBS (pH 7.4), the coverslipswere mounted with glycerol-PBS (1:1 dilution) (pH 8.0), and 200 cellsfor each condition were analyzed in triplicate in blind fashion with aNikon Optiphot fluorescence microscope. The results were expressed interms of percentage of cells showing cytoskeleton rearrangement.

As a negative control, the complex network of F-actin filaments of IEC6cells (stained with fluorescein-phalloidin) were incubated for 24 hrwith 30 μl of pTTQ181 supernatant.

Immunofluorescence localization of ZO-1 within the same cells probedwith anti-ZO-1 antibody 7445, which recognizes both ZO-1 isoenzymes,showed uniform continuous staining of ZO-1 exclusively along thecell-cell boundaries. On the contrary, pZ14 supernatant induced loss ofcentrally located, transcytoplasmatic actin filaments, as wellasreredistribution of ZO-1 from the cell-cell interface. Theredistribution of ZO-1 protein from the tj complex was not associated toits tyrosine phosphorylation.

Taken together, these results indicate that actin polymerization inducedby ZOT is mechanistically linked to the redistribution of the ZO-1protein from the tj complex. Therefore, ZO-1 may be one link betweenZOT-induced actin reorganization and the opening of the tj.

EXAMPLE 2 Intracellular Mediator of ZOT

Several intracellular mediators have been mechanistically linked tochanges in tj permeability, including cAMP, Ca⁺⁺, and PKC (Madara,supra; and Balda et al, J. Membrane Biol., 122:193-202 (1991)). However,the observation that the response to theophylline (a phosphodiesteraseinhibitor) in rabbit ileum pre-exposed to V. cholerae 395 supernatant(containing both CT and ZOT) induced a peak response in Isc similar tothat evoked in tissues exposed to the negative control, suggests thatZOT-induced changes in tissue permeability are not mediated through cAMP(Fasano et al, supra). Similar results were obtained when ZOT-containingsupernatants were tested alone or in combination with purified CT. Theseresults suggest that the total amounts of intracellular cAMP in controlor ZOT-exposed tissues are comparable. Thus, to determine whether PKCmediates ZOT activity, rabbit small intestines and IEC6 cells wereexposed to the PKC inhibitor staurosporine (MacLeod et al, Amer.Physiol. Soc., 192:C950-C955 (1992)).

A. Rabbit Small Intestines

More specifically, rabbit ileum stripped of the muscular and serosallayers was mounted in Ussing chambers, and then exposed to 300 μl (1:33dilution) of pZ14 supernatant, either alone or in combination with 10 nMstaurosporine, added 10 min prior to and throughout the pZ14 supernatantexposure. Changes in Rt where then recorded at 10 min intervals.

10 nM staurosporine was found to completely prevent the decrease of Rtinduced by pZ14 supernatant. Since staurosporine is a potent, butnon-selective inhibitor of PKC activity (Meyer et al, Int. J. Cancer,43:851-856 (1989)), a staurosporine derivative, i.e., CGP41251 thatselectively inhibits PKC (Meyer et al, supra), was then tested onZOT-induced actin reorganization and tissue permeability.

More specifically, rabbit ileum stripped of the muscular and serosallayers was mounted in Ussing chambers and then exposed to 300 μl (1:33dilution) pZ14 supernatant, either alone or in combination with 10 nMCGP41251, added 10 min prior to and throughout the pZ14 supernatantexposure. Ussing chambers pre-exposed to 10 mM of an inactivestaurosporine analogue, i.e., CGP42700 (Meyer et al, supra) for 10 minand throughout the pZ14 supernatant exposure were used as negativecontrols. Changes in Rt where then recorded at 10 min intervals. Theresults are shown in FIG. 1.

As shown in FIG. 1, pre-treatment with 10 nM CGP41251, but not with itsinactive analogue CGP47200, prevented the changes in Rt induced by pZ14supernatant. No significant changes were observed when the tissues wereexposed in a similar manner to pTTQ181 supernatant.

Phorbol esters are a PKC activator. Thus, the phorbol ester12-tetradecanoylphorbol-13-acetate (TPA) was tested to confirm that ZOTacts via PKC.

More specifically, rabbit ileum stripped of the muscular and serosallayers was mounted in Ussing chambers and then exposed to 300 μl (1:33dilution) pZ14 supernatant, either alone or in combination with 10⁻⁸ MTPA added 10 min prior to and throughout the pZ14 supernatant exposure.Changes in Rt where then recorded at 10 min intervals.

A significant reduction in Rt was induced with pZ14 supernatant whencompared to pTTQ181 supernatant (-7.33±2.55 vs -2.57±1.51 mohms.cm² ;p<0.005). When added to the same tissue, TPA together with pZ14supernatant produced a decrease in Rt (total Rt change: -7.37±3.2mohms.cm²) which is not different from that seen with TPA alone. Theseresults demonstrate that the effect of ZOT and TPA on intestinalpermeability is non-additive, suggesting that they both act through PKC.

B. IEC6 Cells

The effect of ZOT on the actin cytoskeleton of IEC6 cells in thepresence of PKC inhibitors was then evaluated.

More specifically, 2.0×10⁵ IEC6 cells were pre-treated with either 10 nMCGP41251 or 10 nM CGP42700 for 30 min prior to and throughout exposureto 30 μof pZ14 supernatant. After 24 hr incubation at 37° C., 2.0×10⁴IEC6 cells/ml, cultured on coverslips, were fixed in 3.7% (v/v)formaldehyde in PBS (pH 7.4) for 10 min at room temperature. Afterwashing in the same buffer, the cells were permeabilized with 0.5% (v/v)Triton X-100 in PBS (pH 7.4) for 10 min at room temperature. The cellswere then incubated with 5.0 μ/ml fluorescein-phalloidin at 37° C. for30 min. Finally, the cells were washed with PBS (pH 7.4), the coverslipswere mounted with glycerol-PBS (1:1 dilution) (pH 8.0), and 200 cellsfor each condition were analyzed in triplicate in blind fashion with aNikon Optiphot fluorescence microscope. The results were expressed interms of the percentage of cells showing cytoskeleton rearrangement.

When pZ14 supernatant was added, 62% of the cells showed a rearrangementof the cytoskeleton, while only 27% of the cells exposed to PBS, and 21%of cells exposed to pTTQ181 supernatants were affected. Pre-treatmentwith 10 nM of CGP41251, but not with 10 nM of its inactive analogueCGP42700, blocked the cytoskeletal effects of ZOT (31% vs 58% of cellsaffected, respectively).

Moreover, ZOT-induced actin polymerization, i.e., a decrease in G- andan increase in F-actin pools was significantly blocked when bovinepulmonary artery endothelial cells were tested as described above, andpre-treated with 10 nM of CGP412512, whereas pre-treatment with 10 nM ofCGP42700 did not cause such blockage (see Table I above).

These combined data indicate that ZOT activates PKC, and that thisactivation occurs proximally to both actin polymerization and finalopening of tj.

C. The PKC State

PKC exists in two states in all cells, i.e., an inactive state in whichthe kinase is associated with the cytoplasm, and an active state inwhich the molecule is translocated from the cytoplasm to the membranes(Thomas et al, Methods Enzymol., 1410:399-435 (1987)). Thus, the stateof PKC was directly measured in IEC6 cells exposed to pZ14 supernatant.

More specifically, confluent IEC6 monolayers were treated with 30 μl ofpZ14 supernatant (1:33 dilution) for increasing time intervals (0, 3min, 10 min). After ZOT exposure, the IEC6 cells were lysed by mechanicdisruption (forced passage through 25 gauge needle 3 times), thecytoplasmic and membrane fractions were separated by centrifugation at13,000×g. for 30 min at 4° C., and assayed for PKC activity bydetermining the incorporation of ³² p from γ-³² ! ATP into histone 1(Ellis et al, Am. J. Physiol. 263:F293-F300 (1992)). As a control, PKCactivity was also determined using a PKC assay system (Gibco BRL, GrandIsland, N.Y.) based on measurement of the phosphorylation of acetylatedmyelin basic protein as described by YasudaCommun. Biochem. Biophys.Res. Commun., 166:1220 (1990). The net PKC activity value reflects thedifference between PKC activity in the presence and absence of PKC19-26, a PKC pseudosubstrate inhibitor (Yasuda et al, supra). Total PKCspecific activity (pmol/min) was normalized to total protein. Theresults are shown in FIGS. 2A-2B.

As shown in FIGS. 2A-2B, pZ14 supernatant induced a significant(1.72-fold) increase of membranous (FIG. 2B), and, to a lesser extend,cytosolic (FIG. 2A), PKC activity. In an average of 3 experiments, thisZOT-induced PKC activity increase peaked at 3 min (134.0 pmol/min/mgprotein vs. baseline value of 77.6 pmol/min/mg protein) and returned tobaseline at 10 min (65.4 pmol/min/mg protein). These data directlydemonstrate that ZOT effect on tissue permeability is mediated by PKC.

D. PKC Isoenzyme

Molecular cloning and sequence analysis of PKC has demonstrated theexistence of a gene family encoding several closely related, butdistinct, isoenzymes with different physiological properties (Azzai etal, Eur. J. Biochem., 208:547-557 (1992)). Staurosporine and its morespecific derivative CGP41251 preferentially inhibit the Ca²⁺ -dependentgroup-A PKC isoenyzmes as compared to the Ca²⁺ -independent group-Bisoenzymes (McGlynn et al, J. Cell Biochem., 49:239-250 (1992); andMarte et al, Cell Growth and Differ., 5:239-247 (1994)). As a result,experiments focusing on PKC-α, the only detectable Ca²⁺ -dependent PKCisoenzyme described in ileal enterocytes (Hyun et al, Comp. Biochem.Physiol., 108C:171-178 (1994)), were carried out.

More specifically, 15-30 μg protein/lane cytosolic membrane fractionsobtained as described above were separated by 8.0% (w/v) SDS-PAGE.Separated proteins were transferred to a nylon membrane (N-Immobilon,Millipore) in a Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). Themembrane was rinsed in PBS containing 0.05% (v/v) Tween 20 (hereinafter"PBS-T"), and blocked in PBS-T containing 5.0% (v/v) non-fat milk for 1hr at room temperature. Affinity-purified anti-PKC-α and anti-PKC-εantibodies (Gibco BRL) were diluted to obtain optimal saturatingconditions (1:500 dilution) in PBS-T containing 0.83% (v/v) non-fatmilk, and incubated with the membrane for 16 hr at 4° C. Followingincubation, the membrane was first washed with 5.0% (v/v) non-fat milkin PBS-T (3× for 15 min), then with PBS-T (1× for 15 min), and incubatedfor 2 hr at room temperature with a 1:30,000 dilution of goatanti-rabbit IgG conjugated to horseradish peroxidase. Followingextensive washing with 5.0% (v/v) non-fat milk in PBS-T, immunoreactivebands were developed using enhanced chemiluminescence (Amersham).

Isoform-specific synthetic peptides (Gibco BRL) having the amino acidsequences for each respective PKC isoform were used as controls. Thesynthetic peptides were based on unique sequences in the

V₃ region of α PKC:(Ala-Gly-Asn-Lys-Val-Ile-Ser-Pro-Ser-Glu-Asp-Arg-Arg-Gln, SEQ ID NO:1),and

V₃ region of ε PKC: (Lys-Gly-Phe-Ser-Tyr-Phe-Gly-Glu-Asp-Leu-Met-Pro,SEQ ID NO:2).

As determined by immunoblotting, acute (3 min) treatment of IEC6 cellswith pZ14 supernatant induced a significant translocation of PKC-αisoenzyme from the cytosol to the membrane of the cells. This reductionwas partially reversed after 10 min of incubation. 10⁻⁷ M TPA induced asimilar, but more sustained (up to 2 hr) reduction of cytosolic PKC-α,whereas down-regulation of this isoform was observed after 24 hrincubation. A continuous, time-dependent accumulation of the PKC-αregulatory subunit was observed in the cellular cytosol. No significantincrease in PKC-α was detected in either membrane fractions obtainedfrom cells exposed to pZ14 supernatant or the TPA positive control.

These results provide strong evidence that PKC-α is the intracellularmediator of ZOT-induced actin reorganization and tj disassembly.

E. Serine Phosphorylation

Experiments in IEC6 cells were also conducted to determine serinephosphorylation of target protein(s) in both cellular membrane orcytosolic subfractions in order to identify the substrate of PKCphosphorylation.

More specifically, 2.0×10⁵ IEC6 cells/wells were exposed to 10⁻¹⁰ Mpurified MBP-ZOT (obtained in Example 5), at increasing time intervals(0, 15 min, 30 min, 45 min). The reaction was stopped with cold PBS (pH7.4) (washed three times), and the cells were scraped and lysed asdescribed above. Cytosolic and membranous subfractions were thenobtained as described above. 20-30 μg of each preparation were separatedby 8.0% (w/v) SDS-PAGE. Separated proteins were transferred to a nylonmembrane (N-Immobilon, Millipore) in a Trans-Blot ElectrophoreticTransfer Cell (Bio-Rad). The membrane was rinsed and blocked in PBS-Tcontaining 5.0% (v/v) non-fat milk for 1 hr at room temperature.Monoclonal anti-serine antibodies (Sigma Immunochemicals) were dilutedto obtain optimal saturating conditions (1:1000 dilution) in PBS-Tcontaining 0.83% (v/v) non-fat milk, and incubated with the membrane for16 hr at 4° C. Following incubation, the membrane was first washed with5.0% (v/v) non-fat milk in PBS-T (3×for 15 min), then with PBS-T (1×for15 min), and incubated for 2 hr at room temperature with a 1:30,000dilution of goat anti-rabbit IgG antibodies conjugated to horseradishperoxidase. Following extensive washing with 5.0% (v/v) non-fat milk inPBS-T, immunoreactive bands were developed using enhancedchemiluminescence (Amersham).

The results showed that purified MBP-ZOT induced time-dependent serinephosphorylation of a cytoplasmic protein of an apparent MW of 100-120kDa. This protein may represent the target of PKC phosphorylationinduced by ZOT, and may be involved in the intracellular signalingleading to the opening of tj.

EXAMPLE 3 Selectivity of ZOT Action

To establish an optimal in vitro system to study the effect of ZOT ontj, several cell lines were screened for ZOT responsiveness. This isbecause the Ussing chamber assay, while sensitive, is not suitable forscreening a large number of samples. In search of an alternative system,a tissue culture assay for ZOT was developed.

A. Specific Effect of ZOT on Different Cell Lines

To establish whether ZOT exerts either a selective or a broad effect ontj regulation, several cell lines were tested for ZOT responsiveness.More specifically, human colon carcinoma cell lines HT-29 Cl 19A (VanDen Berghe et al, Biochem. J., 258:673-679 (1992)), and CaCo2 (Nath etal, J. Diarrhoeal Dis., 8:133-142 (1990)), were grown in cell-cultureflasks (Falcon) under humidified atmosphere of 95% O₂ /5% CO₂ at 37° C.in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal-calfserum, 40 μg/l penicillin and 90 μg/l streptomycin. The cells weresubcultured at a surface ratio of 1:5 after trypsin treatment every 5days, when they had reached 70-80% confluence. The passage number of thecells used in the this study varied between 15 and 30.

The HT-29 Cl 19A or CaCo2 monolayers were grown to confluence (12-14days after plating at a 1:2.5 surface ratio) on tissue-culture-treatedpolycarbonate filters firmly attached to a polystyrene ring (6.4 mmdiameter, Transwell Costar). The filters were placed in a tightlyfitting insert separating the serosal and mucosal compartment of amodified Ussing chamber, and the experiments were carried out asdescribed above for the rabbit intestine.

No significant changes of Rt in HT-29 Cl 19A monolayers (n=4 for up to 3hr each sample tested) exposed to 30 μl of pZ14 supernatant (1:33dilution) were obtained when compared to monolayers exposed to 30 μl ofpTTQ181 supernatant negative control. On the other hand, when tested inCaCo2 cell monolayers, 30 μl of pZ14 supernatant induced a significantdecrease in Rt, suggesting a different susceptibility to ZOT betweenthese two cell lines.

B. F-actin Organization

To study the effect of ZOT on F-actin organization, IEC6 and LLC-PK₁(Hull et al, In Vitro, 12:670-677 (1976)) cell cultures were tested inthe same manner.

The LLC-PK, i.e., pig kidney cortex, cells (Hull et al, supra) weregrown in cell-culture flasks (Falcon) at 37° C. in an atmosphere of 95%O₂ /5% CO₂. The culture medium consisted of Dulbecco's modifiedessential medium supplemented with 10% (v/v) fetal bovine serum. Cellsranging from passage 185 to 200 were passed weekly by trypsinizing with0.25% (w/v) trypsin in 0.02% (w/v) EDTA, when they had reached 70-80%confluent monolayers.

IEC6 cells exposed to 30 μl of pZ14 supernatant, and subsequently probedwith fluorescein-phalloidin, as described above, for F-actin, showedsignificant actin reorganization, while no significant changes weredetected for LLC-PK₁ cells.

The observation that ZOT exerts a selective permeabilizating effect(probably interacting with a specific cellular receptor present only on"sensitive" cells), combined with its effect on cytoskeleton and theactivation of PKC, indicates that ZOT acts via an intracellular pathway,rather then directly on tj.

EXAMPLE 4 Role of Phospholipase C in ZOT Activity

Phospholipase C (hereinafter "PLC") is an enzyme that convertsphosphatidyl inositol diphosphite (hereinafter "PIP₂ ") into inositoltriphosphate (hereinafter "IP₃ ") plus diacylglycerol (hereinafter"DAG"). PLC may participate in the assembly and sealing of ZOs, as wellas in their regulation. The effect of PKC on ZOs is secondary to theactivation of PKC induced by both IP₃ (via Ca²⁺) and DAG (Berridte etal, Nature, 341:197-205 (1989)).

The effect of ZOT on cytoskeletal rearrangement, actin polymerization,and tissue permeability changes has been shown above to involve PKCactivation. To establish whether the primary target of ZOT is PKC orPLC, the experiments in IEC6 cells and Ussing chambers described inExample 1 above were repeated, but the samples were pre-incubated for 10min with 100 mM neomycin sulfate, and throughout the exposure to thepZ14 supernatant. Neomycin is a substance that binds to PIP₂, andprevents its conversion to IP₃ plus DAG by PLC.

IEC6 cell cultures pre-treated with 100 mM neomycin, and then exposed topZ14 supernatant showed a lower percentage of cells presentingcytoskeletal rearrangement (12.0±5.17) as compared to those exposed onlyto ZOT (49.0±9.23; p<0.05). Pre-exposure of rabbit ileum to 100 mMneomycin sulfate partially prevented the increased tissue permeabilityinduced by ZOT in untreated tissues.

These results indicate that the transmembrane PLC may play a role inZOT-induced actin reorganization and tissue permeability.

EXAMPLE 5 Purification of ZOT

5000 ml of pZ14 supernatant was concentrated 1000-fold using a laminaflow filter with a MW cutoff of 10 kDa, and then subjected to 8.0% (w/v)SDS-PAGE. Protein bands were detected by Coomassie blue staining of theSDS-PAGE gel. No protein band corresponding to ZOT was detectable whencompared to control pTTQ181 supernatant treated in the same manner.Therefore, even though the zot gene was placed behind the highlyinducible and strong tac promoter in pZ14, the level of the protein in1000-fold concentrated pZ14 supernatant was still not detectable by theCoomassie stained SDS-PAGE gel.

Hence, to increase the amount of ZOT produced, the zot gene was fused inframe with the maltose binding protein (hereinafter "MBP") gene tocreate a MBP-ZOT fusion protein.

The MBP vector pMAL-c2 (Biolab) was used to express and purify ZOT byfusing the zot gene to the male gene of E. coli. This construct uses thestrong, inducible tac promoter, and the malE translation initiationsignals to give high level expression of the cloned zot gene. The vectorpMAL-c2 has an exact deletion of the male signal sequence, which leadsto cytoplasmic expression of the fusion protein. Affinity chromatographypurification for MBP was used to facilitate isolation of the fusionprotein (Biolab).

More specifically, vector pMAL-c2 was linearized with EcoRI (that cutsat the 3' end of the malE gene), filled in with Klenow fragment, anddigested with XbaI (that has a single site in pMAL-c2 polylinker). Theorf encoding ZOT was subcloned from plasmid pBB241 (Baudry et al,supra). Plasmid pBB241 was digested with BssHII, filled in with Klenowfragment, and digested with XbaI. Then, the blunt-XbaI fragment wassubcloned into pMAL-c2 to give plasmid pLC10-c. Since both the insert,and the vector had blunt and sticky ends, the correct orientation wasobtained with the 3' end of malE fused with the 5' terminus of theinsert. pLC10-c was then electroporated into E. coli strain DH5α. InpBB241, the BssHII restriction site is within the zot orf. Thus, aminoacids 1-8 of ZOT are missing in the MBP-ZOT fusion protein.

In order to purify the MBP-ZOT fusion protein, 10 ml of Luria Bertanibroth containing 0.2% (w/v) glucose and 100 μg/ml ampicillin wereinoculated with a single colony containing pLC10-c, and incubatedovernight at 37° C. with shaking. The culture was diluted 1:100 in 1.0of the same fresh medium, and grown at 37° C. while shaking, to about1.0×10⁸ cells/ml. 0.2 mM IPTG was then added to induce the MBP-ZOTexpression, and the culture was incubated at 37° C. for additional 3 hr.The bacteria were then pelleted and resuspended in 20 ml of ice cold"column buffer" comprising 20 mM Tris-HCl, 0.2 M NaCl, 1.0 mM EDTA, 10mM 2-ME, 1.0 mM NaN₃. The bacterial suspension was lysed by french presstreatment and spun for 30 min at 13,000×g at 4° C. The supernatant wascollected, diluted 1:5 with column buffer and loaded into a 1×10 columnof amylose resin (Biolabs, MBP-fusion purification system),pre-equilibrated with column buffer. After washing the column with 5volumes of column buffer, the MBP-ZOT fusion protein was eluted byloading 10 ml of 10 mM maltose in column buffer. The typical yield from1.0 of culture was 2-3 mg of protein.

The MBP fusion partner of the purified MBP-ZOT fusion protein was thencleaved off using 1.0 μg of Factor Xa protease (Biolabs) per 20 μg ofMBP-ZOT. Factor Xa protease cleaves just before the amino terminus ofZOT. The ZOT protein so obtained was run on a 8.0% (w/v) SDS-PAGE gel,and electroeluted from the gel using an electroseparation chamber(Schleicher & Schuell, Keene, NH).

When tested in Ussing chambers, the resulting purified ZOT induced adose-dependent decrease of Rt, with an ED₅₀ of 7.5×10³¹ 8 M (FIG. 3).

EXAMPLE 6 Production of Anti-ZOT Antiserum

To obtain specific antiserum, a chimeric glutathione S-transferase(GST)-ZOT protein was expressed and purified.

More specifically, oligonucleotide primers were used to amplify the zotorf by polymerase chain reaction (PCR) using plasmid pBB241 (Baudry etal, supra) as template DNA. The forward primer (TCATCACGGC GCGCCAGG, SEQID NO:3) corresponded to nucleotides 15-32 of zot orf, and the reverseprimer (GGAGGTCTAG AATCTGCCCG AT, SEQ ID NO:4) corresponded to the 5'end of ctxA orf. Therefore, amino acids 1-5 of ZOT were missing in theresulting fusion protein. The amplification product was inserted intothe polylinker (SmaI site) located at the end of the GST gene in pGEX-2T(Pharmacia, Milwaukee, WI). pGEX-2T is a fusion-protein expressionvector that expresses a cloned gene as a fusion protein with GST ofSchistosoma japonicum. The fusion gene is under the control of the tacpromoter. Upon induction with IPTG, derepression occurs and GST fusionprotein is expressed.

The resulting recombinant plasmid, named pLC11, was electroporated in E.coli DH5α. In order to purify GST-ZOT fusion protein, 10 ml of LuriaBertani broth containing 100 μg/ml ampicillin were inoculated with asingle colony containing pLC11, and incubated overnight at 37° C. withshaking. The culture was diluted 1:100 in 1.0 of the same fresh mediumand grown at 37° C. while shaking, to about 1.0×10⁸ cells/ml. 0.2 mMIPTG was then added to induce the GST-ZOT expression, and the culturewas incubated at 37° C. for additional 3 hr. The bacteria were thenpelleted, resuspended in 20 ml of ice cold PBS (pH 7.4)and lysed by thefrench press method. The GST-ZOT fusion protein was not soluble underthese conditions as it sedimented with the bacterial pellet fraction.Therefore, the pellet was resuspended in Laemli lysis buffer comprising0.00625 M Tris-HCl (pH 6.8), 0.2 M 2-ME, 2.0% (w/v) SDS, 0.025% (w/v)bromophenol blue and 10% (v/v) glycerol, and subjected toelectrophoresis on a 8.0% (w/v) PAGE-SDS gel, and stained with Coomassiebrilliant blue. A band of about 70 kDa (26 kDa of GST+44 kDA of ZOT),corresponding to the fusion protein, was electroeluted from the gelusing an electroseparation chamber (Schleicher & Schuell, Keene, NH).

10 μg of the resulting eluted protein (10μ20 μg) was injected into arabbit mixed with an equal volume of Freund's complete adjuvant. Twobooster doses were administered with Freund's incomplete adjuvant fourand eight weeks later. One month later the rabbit was bled.

To determine the production of specific antibodies, 10⁻¹⁰ M of ZOT,along with the two fusion proteins MBP-ZOT and GST-ZOT, was transferredonto a nylon membrane and incubated with a 1:5000 dilution of the rabbitantiserum overnight at 4° C. with moderate shaking. The filter was thenwashed 15 min 4 times with PBS-T, and incubated with a 1:30,000 dilutionof goat anti-rabbit IgG conjugated to horseradish peroxidase for 2 hr atroom temperature. The filter was washed again for 15 min 4 times withPBS containing 0.1% (v/v) Tween, and immunoreactive bands were detectedusing enhanced chemiluminescence (Amersham).

On immunoblot, the rabbit antiserum was found and recognize ZOT, as wellas MBP-ZOT and GST-ZOT fusion proteins, but not the MBP negativecontrol.

Moreover, to confirm the production of appropriate anti-ZOT antibodies,neutralization experiments were conducted in Ussing chambers. Whenpre-incubated with pZ14 supernatant at 37° C. for 60 min, theZOT-specific antiserum (1:100 dilution), was able to completelyneutralize the decrease in Rt induced by ZOT on rabbit ileum mounted inUssing chambers.

EXAMPLE 7 Receptor for ZOT

MBP-invasin fusion protein of Yersinia pseudotuberculosis is capable ofbinding to the integrin receptor of mammalian cells, and confers theinvasive phenotype on non-pathogenic E. coli harboring plasmids thatproduce the MBP-invasin fusion protein (Leong et al, The EMBO J.,9(6):1979-1989 (1990)). As a result, experiments were carried out todetermine if the MBP-ZOT fusion protein recognizes a specific intestinalbinding site, as well as retain the ability to increase tissuepermeability.

More specifically, rabbit ileum stripped of the muscular and serosallayers was mounted in Ussing chambers and then exposed to either 10⁻¹⁰ Mpurified MBP-ZOT or purified ZOT, both added to the mucosal side of thetissue. 10⁻¹⁰ M MBP was used as a negative control. Changes in Rt wherethen recorded at 10 min intervals. The results are shown in FIG. 4.

As shown in FIG. 4, purified MBP-ZOT fusion protein (Δ) was found toinduce a Rt decrease in rabbit ileum comparable to that induced bypurified ZOT (□), but significantly different when compared to thechanges induced by the MBP negative control (▪). Both purified MBP-ZOTand ZOT effects on Rt were readily reversible 24 hr following withdrawalof the moieties. These data demonstrate that the ZOT component of theMBP-ZOT fusion protein is still capable of recognizing and binding tothe ZOT intestinal receptor.

At the end of the experiment, the tissues exposed to MBP-ZOT were fixedfor electron microscopy as described by Fasano et al, supra, and thenincubated with gold-labelled anti-MBP monoclonal antibodies (Biolabs NewEngland Lab) (1:25 dilution). Tissues exposed to the MBP-ZOT fusionprotein showed a significant number of immunogold particles.

Similar binding experiments were performed with several cell lines,including IEC6 cells, CaCo₂, T84 (Nath et al, supra), and bovineendothelial cells. 2.0×10⁵ of these cells were incubated at differenttime intervals (5 min, 30 min, 60 min), and temperatures (4° C. or 37°C. with either 5×10⁻⁹ M MBP-ZOT or 5.0×10⁻⁹ M MBP negative control. Thecells were then fixed with cold methanol, and incubated withfluorescein-labelled anti-MBP antibodies (1:100 dilution).

When exposed to the MBP-ZOT fusion protein (at the various temperaturesand time intervals tested) IEC6, CaCo₂, and bovine endothelial cellmonolayers, displayed a significant increased number of fluorescentparticles as compared to cells exposed to the MBP negative control. Onthe contrary, no significant staining was observed in T84 cells whenincubated with MBP-ZOT. These results suggest that ZOT interacts with aspecific surface receptor whose distribution among different cell linesvaries.

The same type of experiments were repeated using IEC6 monolayers exposedfor 60 min at 4° C. to 10⁻¹⁰ M of the MBP-ZOT fusion protein, and thenincubated with a 1:500 dilution of the anti-ZOT antiserum. Again, cellsexposed to the MBP-ZOT fusion protein (at the same time intervals andtemperatures described above) showed a significant number of fluorescentparticles as compared to the MBP negative control, confirming that theligand consisted of the fusion protein, and not a degradation productcontaining MPB, but not ZOT.

Similar results were obtained when using purified ZOT, and the same celllines and experimental conditions tested above, and incubating the cellmonolayers with fluorescein-labelled anti-ZOT antiserum.

To establish the regional distribution of the ZOT receptor within theintestine and along the villous-crypt axis, different intestinalsegments, including jejunum, proximal and distal ileum, and colon, weremounted in Ussing chambers, and exposed to either 10⁻⁹ M purifiedMBP-ZOT fusion protein or 10⁻⁹ M MBP negative control.

When using the purified MBP-ZOT fusion protein, significant reduction ofRt was observed in the jejunum and distal ileum, while the colonremained unaffected. Once the maximal effect of the MBP-ZOT fusionprotein on Rt was obtained, the tissues exposed to either MBP-ZOT or MBPwere fixed, and stained as described above.

The segments that showed a decrease in Rt, i.e., jejunum and distalileum, displayed a significant increased number of fluorescent particlescompared to the same tract of intestine exposed to the MBP negativecontrol.

The data demonstrates that distribution of the ZOT receptor varieswithin the intestine, being more represented in the jejunum and distalileum, and decreases along the villous-crypt axis. This distributioncoincides with the preferential F-actin redistribution induced by ZOT inthe mature cells of the villi.

The results in Examples 1-7 above demonstrate that (1) ZOT interactswith a cell membrane receptor, whose surface expression among variouscells, and within the intestine varies, i.e., such is present in thejejunum and distal ileum, but not in the colon; it decreases along thevillous-crypt axis; (2) ZOT induces time-and dose-dependentrearrangement of the cytoskeleton; (3) this rearrangement is related tothe PKC-α -dependent polymerization of actin monomers into actinfilaments and is mechanistically linked to the redistribution of theZO-1 protein from the tight junctional complex; (4) this intracellularsignaling leads to the reversible opening of the tj, and is probablyoperating during the physiologic regulation of the paracellular pathway.

EXAMPLE 8 ZOT as an Intestinal Absorption Enhancer

The observation that ZOT (1) does not affect the viability of theintestinal epithelium, (2) is not cytotoxic, (3) fails to completelyabolish the intestinal transepithelial resistance, and, mostimportantly, (4) induces a reversible increase of tissue permeability,indicated in the present invention that ZOT is useful a tool to modulatethe intestinal tj so as to enhance absorption of biologically activeingredients. This was confirmed by the following in vitro and in vivostudies.

A. In vitro Studies

2-3 kg adult male New Zealand white rabbits were sacrificed by cervicaldislocation. A 20 cm segment of distal ileum was excised, opened alongthe mesenteric border, and rinsed free of intestinal content. Eightpieces of intestine stripped of the serosal and muscular layers werethen mounted in Ussing chambers (1.12 cm² opening), and bathed byfreshly prepared Ringer's solution. The solution was kept at 37° C., andgassed with 95% O₂ /5% CO₂ throughout the experiment.

As prototype biologically active ingredients to be delivered, insulinlabelled with ¹²⁵ I, and IgG labelled with ¹²⁵ I were tested.

Once the tissues reached a steady state condition, paired tissues,matched on the basis of their resistance, were exposed luminally toeither 10⁻¹¹ M insulin or 156.25-ng IgG, alone or in the presence of10⁻¹⁰ M MBP-ZOT fusion protein. Then, 1.0 ml from the serosal side and50 μgl from the mucosal side were immediately obtained to establishbaseline values. Samples from the serosal sides were then collected at20 min intervals for the following 80 min. The reservoirs were thenemptied, washed twice with Ringer's solution, and refilled with freshRinger's solution containing only 10⁻¹¹ M insulin or 156.25 ng IgGpreviously added to the chamber. The results are shown in FIGS. 5A and5B.

As shown in FIGS. 5A and 5B, the MBP-ZOT fusion protein was found toincrease, in a time-dependent and reversible manner, the transintestinalabsorption of both insulin (2-fold) (FIG. 5A) and IgG (10-fold) (FIG.5B). These changes paralleled a decrease in the intestinal Rt.

When purified ZOT was tested in the same manner in place of the MBP-ZOTfusion protein, a time-dependent and reversible transintestinalabsorptive increase of both insulin (2-fold) (FIG. 6A) and IgG (+50%)(FIG. 6B) was also observed. This increase in absorption coincided withthe reduction of Rt induced by purified ZOT.

B. In vivo Studies

The intestinal perfusion assay was used as an in vivo test to establishthe effect of ZOT on the translocation of molecules from the intestinallumen to the bloodstream. Intestinal perfusion was carried out accordingto that described by Sladen et al, Biochem. Biophys. Acta, 288:443-456(1972) with minor modifications, i.e., rabbits rather then rats wereemployed, and a flow rate 0.4 ml/min rather then 0.2 ml/min wasemployed, as well as cannulation of the mesenteric veins draining theperfused segments.

More specifically, after a 24 hr fast, 2.5-3 kg adult male New Zealandwhite rabbits were anesthetized with 50 mg ketamine per kg body weight,followed by intramuscularly injection of 7.0 mg xylazine per kg bodyweight. Their body temperature was kept at 37° C. by a lamp. Theabdominal cavity was opened by a midline incision and three distinctsegments of the intestine: (1) the proximal jejunum below the ligamentof Treitz, (2) the distal ileum, and (3) the proximal colon, werecannulated. A second cannula was placed 10-15 cm below the proximalcannula. The segments were rinsed free of intestinal contents with 0.9%(w/v) NaCl warmed to 37° C. The proximal cannulae were connected by apolyvinyl tube to a peristaltic pump (model WPI SP220 I), and the 3segments were perfused at a rate of 0.4 ml/min with a solutioncomprising 2.0 mmol/l glucose, 4.0 mmol/l KCl, 25 mmol/l NaHCO₃, 3.0 g/lPEG-4000 and 3.0 mCi/100 ml ¹⁴ C-PEG-4000. This solution also containedeither 143 pmol/ml of insulin or 8.33 ng/ml of IgG. All of the solutionswere made isotonic by adjustment with NaCl, and the pH fixed at 7.4 bygassing with 95% O₂ /5% CO₂.

Eluates were collected in 20 min aliquots from the distal cannulae. Aninitial equilibrium period of 30 min was allowed, followed by threeconsecutive 20 min collection periods for baseline measurement of thenet transport of the biologically active ingredient tested, along withwater and electrolytes, in each segment studied.

Subsequently, 10⁻¹⁰ M ZOT in PBS containing either 143 pmol/ml ofinsulin or 8.33 ng/ml of IgG, was added to the perfusion solution, and asecond perfusion period (30 min equilibration+3×20 min collection) wascarried out.

To establish whether the effect of ZOT on intestinal permeability wasreversible, a third perfusion period was performed with the same PBSused in the first period.

At the end of each perfusion period, the mesenteric vein draining eachsegment perfused was cannulated, and a blood sample was obtained tomeasure the amount of biologically active ingredient and PEG thatreached the bloodstream. At the end of the experiment, the animal wassacrificed and the segments perfused were isolated, measured, dried andweighed. The results are shown in FIGS. 7A-7C.

As shown in FIGS. 7A-7C, purified ZOT induced a 10-fold increase of thepassage of insulin (▴) both in the jejunum (FIG. 7A) and distal ileum(FIG. 7B), while no substantial changes were observed in the colon (FIG.7C). The increased absorption of insulin induced by ZOT paralleled thereduction of water absorption (□) following the opening of tj. Thiseffect was completely reversible within 60 min of the withdrawal of ZOTfrom the perfusion solution.

Purified ZOT also significantly increased the blood concentration ofboth insulin (open bar) and the non-absorbable marker PEG-4000 (shadedbar) in the jejunum and ileum, but not in the colon (FIG. 8). The amountof insulin and PEG-4000 returned to the baseline values once ZOT waswithdrawn.

Similar results were observed with IgG, i.e., ZOT induced an increase ofthe transintestinal absorption of IgG (∘) 6-fold in the jejunum (FIG.9A) and 2-fold in the ileum (FIG. 9B). No significant changes weredetected in the colon (FIG. 9C). The increased IgG absorption wasreversible, and coincided with reduction of water absorption (□) inducedby ZOT.

Purified ZOT also increased the blood concentration of both IgG (2-fold)(open bar) and PEG-4000 (+75%) (shaded bar) in the mesenteric veindraining the small intestinal segments perfused, while no changes wereobserved in the bloodstream of the colonic segment perfused (FIG. 10).

The above results demonstrate that co-administration of a biologicallyactive ingredient with ZOT enhances intestinal delivery of thebiologically active ingredient, and that this enhancement is effectivefor both relatively small molecules (5733 Da: insulin) and largemolecules (140-160 kDA: IgG).

While the invention has been described in detail, and with reference tospecific embodiments thereof, it will be apparent to one of ordinaryskill in the art that various changes and modifications can be madetherein without departing from the spirit and scope thereof.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic peptide                                         (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AlaGlyAsnLysValIleSerProSerGluAspArgArgGln                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic peptide                                         (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       LysGlyPheSerTyrPheGlyGluAspLeuMetPro                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TCATCACGGCGCGCCAGG18                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic DNA                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GGAGGTCTAGAATCTGCCCGAT22                                                      __________________________________________________________________________

What is claimed:
 1. An oral dosage composition for intestinal deliveryof a therapeutic agent comprising:(A) a therapeutic agent selected fromthe group consisting of a drug compound, a biologically active peptideand a vaccine; and (B) an intestinal absorption enhancing effectiveamount of purified Vibrio cholera zonula occludens toxin.
 2. The oraldosage composition of claim 1, wherein said drug compound is selectedfrom the group consisting of a drug which acts on the cardiovascularsystem, a drug which acts on the central nervous system, anantineoplastic drug and antibiotics.
 3. The oral dosage composition ofclaim 2, wherein said drug which acts on the cardiovascular system isselected from the group consisting of lidocaine, adenosine, dobutamine,dopamine, epinephrine, norepinephrine and phentolamine.
 4. The oraldosage composition of claim 2, wherein said drug which acts on thecentral nervous system is selected from the group consisting ofdoxapram, alfentanil, dezocin, nalbuphine, buprenorphine, naloxone,ketorolac, midazolam, propofol, metacurine, mivacurium andsuccinylcholine.
 5. The oral dosage composition of claim 2, wherein saidantineoplastic drug is selected from the group consisting of cytarabine,mitomycin, doxorubicin, vincristine and vinblastine.
 6. The oral dosagecomposition of claim 2, wherein said antibiotic is selected from thegroup consisting of methicillin, mezlocillin, piperacillin, cetoxitin,cefonicid, cefmetazole and aztreonam.
 7. The oral dosage composition ofclaim 1, wherein said biologically active peptide is selected from thegroup consisting of a hormone, lymphokine, globulin and albumin.
 8. Theoral dosage composition of claim 7, wherein said hormone is selectedfrom the group consisting of testosterone, nandrolene, menotropins,insulin and urofolltropin.
 9. The oral dosage composition of claim 7,wherein said lymphokine is selected from the group consisting ofinterferon-α, interferon-β, interferon-γ, interleukin-1, interleukin-2,interleukin-4 and interleukin-8.
 10. The oral dosage composition ofclaim 7, wherein said globulin is an immunoglobulin selected from thegroup consisting of polyvalent IgG, specific IgG, specific IgA andspecific IgM.
 11. The oral dosage composition of claim 1, wherein theratio of therapeutic agent to zonula occludens toxin is in the range ofabout 1:10 to 3:1.
 12. The oral dosage composition of claim 11, whereinthe ratio of therapeutic agent to zonula occludens toxin is in the rangeof about 1:5 to 2:1.
 13. The oral dosage composition of claim 1, whereinzonula occludens toxin is present in the composition in an amount offrom about 40 ng to 1000 ng.
 14. The oral dosage composition of claim13, wherein zonula occludens toxin is present in the composition in anamount of from about 400 ng to 800 ng.
 15. A method comprising orallyadministering an oral dosage composition for intestinal delivery of atherapeutic agent comprising:(A) a therapeutic agent selected from thegroup consisting of a drug compound, a biologically active peptide and avaccine; and (B) an intestinal absorption enhancing effective amount ofpurified Vibrio cholera zonula occludens toxin.
 16. The method of claim15, wherein said drug compound is selected from the group consisting ofa drug which acts on the cardiovascular system, a drug which acts on thecentral nervous system, an antineoplastic drug and antibiotics.
 17. Themethod of claim 16, wherein said drug which acts on the cardiovascularsystem is selected from the group consisting of lidocaine, adenosine,dobutamine, dopamine, epinephrine, norepinephrine and phentolamine. 18.The method of claim 16, wherein said drug which acts on the centralnervous system is selected from the group consisting of doxapram,alfentanil, dezocin, nalbuphine, buprenorphine, naloxone, ketorolac,midazolam, propofol, metacurine, mivacurium and succinylcholine.
 19. Themethod of claim 16, wherein said antineoplastic drug is selected fromthe group consisting of cytarabine, mitomycin, doxorubicin, vincristineand vinblastine.
 20. The method of claim 16, wherein said antibiotic isselected from the group consisting of methicillin, mezlocillin,piperacillin, cetoxitin, cefonicid, cefmetazole and aztreonam.
 21. Themethod of claim 15, wherein said biologically active peptide is selectedfrom the group consisting of a hormone, lymphokine, globulin andalbumin.
 22. The method of claim 21, wherein said hormone is selectedfrom the group consisting of testosterone, nandrolene, menotropins,insulin and urofolltropin.
 23. The method of claim 21, wherein saidlymphokine is selected from the group consisting of interferon-α,interferon-β, interferon-γ, interleukin-1, interleukin-2, interleukin-4and interleukin-8.
 24. The method of claim 21, wherein said globulin isan immunoglobulin selected from the group consisting of polyvalent IgG,specific IgG, specific IgA and specific IgM.
 25. The method of claim 15,wherein the ratio of therapeutic agent to zonula occludens toxin is inthe range of about 1:10 to 3:1.
 26. The method of claim 25, wherein theratio of therapeutic agent to zonula occludens toxin is in the range ofabout 1:5 to 2:1.
 27. The method of claim 15, wherein zonula occludenstoxin is present in the composition in an amount of from about 40 ng to1000 ng.
 28. The method of claim 27, wherein zonula occludens toxin ispresent in the composition in an amount of from about 400 ng to 800 ng.29. The method of claim 15, wherein the zonula occludens toxin isadministered in an amount such that the final concentration in theintestine is in the range of about 10⁻⁵ M to 10⁻¹⁰ M.
 30. The method ofclaim 29, wherein the zonula occludens toxin is administered in anamount such that the final concentration in the intestine is in therange of about 10⁻⁶ M to 5.0×10⁻⁸ M.